Mycobacterium ulcerans and Mycobacterium marinum are pathogenic mycobacteria which cause persistent cutaneous lesions in humans. M. marinum, like M. tuberculosis is a facultative intracellular pathogen which causes a granulomatous skin disease in humans, and life threatening systemic infections in immunocompromised patients. Like M. tuberculosis, M. marinum replicates within macrophages in an intracellular compartment which shows arrested maturation. Work in our laboratory is directed towards understanding how M. marinum commandeers the macrophage for its own purposes and avoids degradation via the lysosomal pathway. Because of the close genetic relationship between M. marinum and M. tuberculosis, we hypothesize that the mechanisms involved in intramacrophage relication will be similar if not identical. Using fusions to Green Fluorescent Protein, we have isolated several genes from M. marinum expressed differentially in macrophages and have shown that close homologues are present in M. tuberculosis. Once the role of these genes in pathogenesis is established, we will make mutations in the M. tuberculosis homologues. The overall goal of this work is to understand how intracellular mycobacteria are able to survive and replicate within phagocytic cells.M. ulcerans causes a severe necrotic skin lesion (Buruli ulcer) remarkable for the absence of an acute inflammatory response in the presence of extensive tissue damage. We have isolated a toxin from M. ulcerans, amd shown with both biochemical and genetic evidence that this molecule plays a major role in the pathogenesis of Buruli ulcer. The toxin molecule, mycolactone, is a 12-membered ring macrolide which at picogram quantities places eucaryotic cells in cell cycle arrest (G1) followed by death via apoptosis. We are currently investigating the precise mechansm by which mycolactone induces cell damage and immunosuppression. In addition we are expoloring whether a toxin minus mutant, might be an effective vaccine. Although macrolides are lipophilic, and thus not antigenic by themselves, we are exploring the possibility of making this molecule antigenic and determining whether an anti-toxin might be valuable as a therapeutic agent for Buruli ulcer. Finally, the exquisite and differential sensitivity of some tumor cell lines suggests this molecule may have value as an anti-neoplastic agent. We are exploring this possibility.